臂旁核谷氨酸能神经元调控睡眠-觉醒周期的作用及神经环路机制

The parabrachial nucleus (PB) is located in the dorsal lateral pons. The patients with the PB lesion or hemorrhage in the PB region showed serious sleepiness, even caused the consciousness dysfunction and coma. These findings indicated that PB plays an essential role in regulating sleep-wake cycle. However, the roles and mechanisms of neuronal pathway of PB glutamatergic neurons in sleep-wake regulation remain to be elucidated. PB glutamatergic neurons send dense projections to the wake related nuclei including locus coeruleus, raphe nucleus, basal forebrain, hypothalamus, and cerebral cortex. We hypothesize that activation of PB glutamatergic neurons can excite wake-related nuclei to induce wakefulness, and on the other hand, inhibition of PB glutamatergic neurons can silence the wake-related nuclei to maintain sleep. In the proposed project, we are going to use (vesicle glutamate 2 transporter, vGlut2)-Cre mice for glutamatergic neurons, the pharmacogenetics and optogenetics to control the neuron activity, to excite or inhibit the activity of PB glutamategic neurons in freely moving mice artificially , combined with automatic sleep bioassay systems, brain-slice patch clamp, and histochemistry approaches, we try to clarify the key role of PB glutamatergic neurons in regulating sleep-wake cycle, and the neuronal circuits involved. The outcome of the proposal will provide new insights into sleep-wake regulation. This project will shed light on the mechanisms underlying the sleep-wake regulation, aid in the development of novel wake promoting drugs, and help to treat the patients with sleep disorders including sleepiness.

臂旁核（parabrachial nucleus，PB）位于脑桥喙部背外侧，临床发现其损伤或内出血的患者常伴有嗜睡，甚至意识障碍、昏迷，提示PB可能对睡眠-觉醒行为调控至关重要，但其调节睡眠-觉醒的作用及神经环路机制仍不明了。PB神经元主要为谷氨酸能神经元，向脑内觉醒核团蓝斑、中缝核、基底前脑、下丘脑和大脑皮层等有密集的纤维投射。因此，我们假说激活PB谷氨酸能神经元可兴奋觉醒核团诱发觉醒，抑制PB谷氨酸能神经元可沉默觉醒核团维持睡眠。本题将借助囊泡膜谷氨酸转运体2（vGlut2）-Cre小鼠，利用能特异性控制神经元活性的药理遗传学和光遗传学技术，人工控制PB谷氨酸能神经元活性，结合自动化睡眠觉醒解析平台、脑片膜片钳、分子生物学及组织学等手段，阐明PB谷氨酸能神经元调控睡眠-觉醒的关键作用及其神经环路机制。旨在丰富和发展睡眠-觉醒调节理论，提供促醒药物新靶点，为临床治疗嗜睡等提供理论基础。

临床发现臂旁核（parabrachial nucleus，PB）损伤可导致患者嗜睡，甚至意识障碍及昏迷，本研究拟揭示PB谷氨酸能神经调控睡眠与觉醒的作用和神经功能环路。借助脑立体定位法将腺相关病毒（AAV）携带的人源胆碱能受体基因载体（hM3Dq）或光通道蛋白（ChR2）微量（200 nl/侧）注射至大鼠双侧PB。脑片膜片钳技术检测hM3Dq或ChR2控制大鼠PB谷氨酸能神经元的作用及效率。对PB表达hM3Dq或ChR2的大鼠，安置脑电/肌电记录电极，在体调控大鼠PB谷氨酸能神经元活性并结合睡眠-觉醒行为记录。研究发现标记蛋白mCherry提示hM3Dq或ChR2在大鼠PB谷氨酸能神经元表达。脑片膜片钳下灌流500 nM氧化氯氮平（CNO）5 min可激活大鼠PB谷氨酸能神经元hM3Dq，使神经元持续放电显著增加。腹腔注射剂量为0.3 mg/kg的CNO可引起PB谷氨酸能神经元表达hM3Dq的大鼠持续觉醒长达15 h，且不伴有睡眠反弹。进一步发现只有激活PB内侧部（MPB）谷氨酸能神经元才能引起长时间的觉醒，而激活PB外侧部谷氨酸能神经元无效。脑片膜片钳技术检测发现表达ChR2的大鼠PB谷氨酸能神经元，给予频率1、5、10、20或40 Hz，持续时间5 ms的蓝光（波长473 nm）刺激模式均能引起该神经元爆发动作电位。提示hM3Dq及ChR2均能控制PB谷氨酸能神经元活性。基础状态下，PB谷氨酸能神经元表达hM3Dq的大鼠与对照组大鼠的睡眠-觉醒行为无显著差异。光遗传学结合睡眠-觉醒行为记录发现，蓝光（频率分别为1、5、10、20或40 Hz，持续时间5 ms）在体激活大鼠PB谷氨酸能神经元能引起大鼠觉醒，觉醒潜伏期约5～10 s。蓝光（频率为20或40 Hz，持续时间5 ms）在体分别刺激外侧下丘脑和基底前脑的PB谷氨酸能神经末梢，调节MPB-外侧下丘脑或MPB-基底前脑神经通路，可引起大鼠脑电δ波幅度降低和觉醒行为。本研究发现MPB谷氨酸能神经元通过调节外侧下丘脑和基底前脑神经环路启动和维持觉醒。